It is now well known that cells can be infected by HIV through a process by which fusion occurs between the cellular membrane and the viral membrane. The generally accepted model of this process is that the viral envelope glycoprotein complex (gp120/gp41) interact with cell surface receptors on the membranes of the target cells. Following binding of gp120 to cellular receptors (e.g., CD4 in combination with a chemokine co-receptor such as CCR-5 or CXCR-4), induced is a conformational change in the gp120/gp41 complex that allows gp41 to insert into the membrane of the target cell and mediate membrane fusion.
The amino acid sequence of gp41, and its variation among different strains of HIV, are well known. FIG. 1 is a schematic representation of the generally accepted functional domains of gp41 (note the amino acid sequence numbers may vary slightly depending on the HIV strain). The fusion peptide (fusogenic domain) is believed to be involved in insertion into and disruption of the target cell membrane. The transmembrane domain, containing the transmembrane anchor sequence, is located at the C-terminal end of the protein. Between the fusion peptide and transmembrane anchor are two distinct regions, known as heptad repeat (HR) regions, each region having a plurality of heptads. The HR1 region, nearer to the N-terminal end of the protein than the HR2 region, has been generally described as comprising the amino acid sequence having the sequence of SEQ ID NO:1. However, due to naturally occurring polymorphisms, the amino acid sequence (and also numbering of residues) of the HR1 region of HIV-1 gp41 may vary, depending on the viral strain from which the amino acid sequence was deduced. The other region, HR2, also depicted in FIG. 1 and SEQ ID NO:2, can also vary with polymorphisms thereof. The amino acid sequence comprising the HR1 region and the amino acid sequence comprising the HR2 region are each one of the most highly conserved regions in the HIV-1 envelope protein (Shu et al., 1999, Biochemistry, 38:5378-5385; Hanna et al., 2002, AIDS 16:1603-8). The HR regions have a plurality of 7 amino acid residue stretches or “heptads” (the 7 amino acids in each heptad designated “a” through “g”), wherein the amino acids in the “a” position and “d” position are generally hydrophobic. Also present in each HR region is one or more leucine zipper-like motifs (also referred to as “leucine zipper-like repeats”) comprising an 8 amino acid sequence initiating with, and ending with, an isoleucine or leucine. Most frequently, the HR2 region has just one leucine zipper like-motif, whereas the HR1 region has five leucine zipper-like motifs. Heptads and leucine zipper-like motifs contribute to formation of a coiled coil structure of gp41 and of a coiled coil structure of peptides derived from the HR regions. Generally, coiled coils are known to be comprised of two or more helices that wrap around each other in forming oligomers, with the hallmark of coiled coils being a heptad repeat of amino acids with a predominance of hydrophobic residues at the first (“a”) and fourth (“d”) positions, charged residues frequently at the fifth (“e”) and seventh (“g”) positions, and with the amino acids in the “a” position and “d” position being determinants that influence the oligomeric state and strand orientation (see, e.g., Akey et al., 2001, Biochemistry, 40:6352-60).
It was discovered that synthetic peptides derived from either the HR1 region (“HR1 peptides”) or HR2 region (“HR2 peptides”) of HIV gp41 inhibit transmission of HIV to host cells both in in vitro assays and in in vivo clinical studies (see, e.g., Wild et al., 1994, Proc. Natl. Acad. Sci. USA, 91:9770-9774; U.S. Pat. Nos. 5,464,933 and 5,656,480 licensed to the present assignee; and Kilby et al., 1998, Nature Med. 4:1302-1306). More particularly, HR1 peptides as exemplified by DP107 (also known as T-21; SEQ ID NO:3) blocked infection of T cells with 50% effective concentration values (EC50) of 1 μg/ml (see, e.g., Lawless et al., 1996, Biochemistry, 35:13697-13708). HR2 peptides, as exemplified by DP178 (also known as T-20; SEQ ID NO:4) typically blocked infection of T cells with 50% effective concentration values (EC50) in the ng/ml range. Pioneering potent synthetic peptides, which comprise one or more enhancer sequences linked to a core HIV gp41 amino acid sequence, inhibit HIV membrane fusion, thereby preventing transmission of the virus to a host cell, have been described previously (see, e.g., U.S. Pat. Nos. 6,258,782 and 6,348,568 assigned to the present assignee). However, HIV gp41-derived synthetic peptides have a relatively low molecular weight. Like other peptides known in the art, in order to be effective as therapeutic agents, such synthetic peptides must be administered frequently (e.g., daily injections) to attain and maintain a level in the bloodstream sufficient for a therapeutic effect. In efforts to overcome this limitation, researchers have attempted to chemically modify a therapeutic agent, such as a peptide or peptidomimetic, by, for example, linking the therapeutic agent to a water-soluble polymer such as polyethylene glycol (PEG) so as to enable the therapeutic agent to survive longer in vivo (e.g., to increase the half-life in the bloodstream and/or to inhibit degradation of the therapeutic agent in the bloodstream). However, as known to those skilled in the art (see, e.g., U.S. Pat. Nos. 6,258,774 and 6,113,906), such modifications to the therapeutic agent have inherent limitations, i.e. such modifications typically limit the bioavailability of the therapeutic agent. More particularly, attaching a water-soluble polymer to a therapeutic agent, particularly a small peptide, frequently modulates the biological activity of the therapeutic agent in a deleterious manner. This loss of both activity and therapeutic usefulness is often the case with lower molecular weight (e.g., less than 4,000 daltons) peptides which have few attachment sites not associated with bioactivity. While the prior art may teach conjugating therapeutic agents to a water-soluble polymer, the prior art fails to teach a conjugate comprising a polymer attached to two or more molecules of synthetic peptide, wherein the conjugate retains substantial bioactivity (e.g., retains substantial biological activity as compared to synthetic peptide alone), and durability (substantial biological activity against a strain of HIV-1 resistant to a synthetic peptide not in the form of a conjugate, as compared to that of the synthetic peptide).
Thus, there is a need for conjugates which can interfere with the interaction of the various domains of gp41 involved in the viral fusion process, and more preferably with the conformational changes of gp41 necessary to effect fusion, thereby inhibiting the fusion of HIV gp41 to a target cell membrane. Additionally, there is a need for conjugates that can inhibit transmission of HIV to a target cell, while retaining substantial biological activity and exhibiting durability. The present invention addresses these needs.